The hepatitis C virus (HCV) causes one of the world's most pandemic and insidious diseases. According to the World Health Organization, there are approximately 170 million carriers worldwide with prevalence up to 0.5-10% [Release, Lancet, 351: 1415 (1998)]. In the United States, four million individuals are afflicted with hepatitis C [Alter and Mast, Gastroenterol Clin North Am, 23: 437-455 (1994)], of which 75% to 85% will develop a chronic infection. This may ultimately lead to cirrhosis (10% to 20%) and hepatocellular carcinoma (1% to 5%) [Cohen, Science, 285: 26-30 (1999)]. The causative agent, HCV, was identified in 1989 and accounted for 50% to 60% of the non-A, non-B transfusion associated hepatitis [Alter et al., N Engl J Med, 321: 1494-1500 (1989); Choo et al., Science, 244: 359-362 (1989); Kuo et al., Science, 244: 362-364 (1989)]. More than 100 strains of the virus have been identified, and are grouped into six major genotypes which tend to cluster in different regions of the world [Simmonds, Current Studies in Hematology and Blood Transfusion, Reesink, ed., Karger, Basel, pp. 12-35 (1994); van Doorn, J Med Vir, 43: 345-356 (1994)].
To date, interferon-alpha monotherapy and interferon-alpha-2b and ribavirin combination therapy (REBETRON® (combination therapy containing REBETOL® (ribavirin, USP) capsules and INTRON® A (interferon alpha-2b, recombinant) injection) Schering-Plough, Kenilworth, N.J.) are the only approved treatments. However, in one study less than 10% of the patients responded to interferon-alpha monotherapy and 41% of the patients responded to REBETRON® (combination therapy containing REBETOL® (ribavirin, USP) capsules and INTRON® A (interferon alpha-2b, recombinant) injection) [Reichard et al., Lancet, 351: 83-87 (1998)]. The most promising antiviral targets in chronic HCV infection are the replication enzymes, RNA-binding proteins, viral entry proteins and enzymes required for viral maturation. Therefore, it would be advantageous if those skilled in the art had the means to develop more effective antiviral agents against the various viral targets to effectively combat this disease.
HCV is a member of the Flaviviridae family. It is a positive-sense, single-stranded RNA virus with genome size of approximately 9.4 kb [Heinz, Arch Viral Supp, 4: 163-171 (1992); Mizokami and Ohba, Gastroenterol JPN, 28 Supp 5: 42-44 (1993); Ohba et al., FEBS Lett, 378: 232-234 (1996); Takamizawa et al., J Virol, 65: 1105-1113 (1991)]. HCV genomic RNA encodes a polyprotein of approximately 3000 amino acid residues: NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH [Lohmann et al., J Hepatol, 24: 11-19 (1996); Simmonds, Clin Ther, 18 Supp B: 9-36 (1996)]. The polyprotein undergoes subsequent proteolysis by host and viral enzymes to yield mature viral proteins [Grakoui et al., J Virol, 67: 1385-1395 (1993); Shimotohno et al., J Hepatol, 22: 87-92 (1995)].
The NS3 protein has been the target of interest for antiviral discovery because of its important roles in HCV maturation and replication. There are two major functional domains: the amino-terminal one third of the protein is a serine protease responsible for certain key aspects of polyprotein processing [Shimotohno et al., J Hepatol, 22: 87-92 (1995)], and the carboxy-terminal two thirds shares sequence similarity with the DEAD box family of RNA helicases [Gorbalenya et al., FEBS Lett, 235: 16-24 (1988); Koonin and Dolja, Crit. Rev Biochem Mol, 28: 375-430 (1993); Korolev et al., Protein Science, 7: 605-610 (1998)].
RNA helicases are grouped into two major superfamilies (SFI and SFII) on the basis of the occurrence of seven conserved motifs, a smaller superfamily (SFIII), and two smaller families [Gorbalenya and Koonin, Curr Opin Struct Biol, 3: 419-429 (1993)]. RNA helicases are mostly of the SFII superfamily and can be further classified into families on the basis of particular consensus sequences in the conserved motifs [de la Cruz et al., TIBS, 24: 192-198 (1999)]. The HCV NS3 RNA helicase is classified as a DExH protein of the SFII superfamily. HCV helicase has two enzymatic activities: NTPase, which is believed to provide an energy source for the unwinding reaction through NTP hydrolysis, and nucleic acid unwinding [Kim et al., Virus Res, 49: 17-25 (1997); Suzich et al., J Virol, 67: 6152-6158 (1993)]. As such, HCV RNA helicase is essential for replication and production of infectious virions, which makes it an excellent target for therapeutics [Kadaré and Haenni, J Virol, 71: 2583-2590 (1997)]. Studies of the crystal structure of HCV helicase reveal that it has three subdomains: subdomain I, which contains NTP and Mg++ binding sites; subdomain II, which is believed to contain a nucleic acid binding site; and subdomain III, which has an extensive helical structure. A coupling region lies between subdomains I and II, and is believed to be involved in transforming chemical energy into motion associated with unwinding [Kim et al., Structure, 156: 89-100 (1998); Cho et al., JBC, 273: 15045-15052 (1998); Yao et al., Nat StructBiol, 4: 463-467 (1997)]. The functions of some of these motifs have been elucidated by studies of the effects of mutations on NTP and RNA binding, NTP hydrolysis and unwinding activity [Pause and Sonenberg, Curr Opin Struct Biol, 3: 953-959 (1993)]. Recently, the basic mechanism for RNA duplex unwinding by the DExH RNA helicase NPH-II was described [Jankowsky et al., Nature, 403: 447-451 (2000)], however, in almost all cases the precise mechanism and the substrates of these enzymes have not been defined. Therefore, it would be beneficial to those skilled in the art to have suitable fragments of the HCV NS3 helicase which could be used to provide such valuable information and simplify the development of specific inhibitors for this enzyme. Nevertheless, there has been no report of an HCV helicase subdomain or fragment that is suitable for this purpose.
To better study the enzymatic properties of the HCV NS3 helicase (e.g., NTP binding, single and double stranded nucleic acid binding sites, energy coupling and helicase activity) and develop potential inhibitors against this enzyme, it is desirable to have suitable fragments of the protein for use in methods or techniques such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. For example, recent developments in NMR-based drug discovery methods provide a powerful means for identifying and optimizing non-peptide drug-like leads, however, such methods are currently limited to proteins having a size of about less than 30 kDa [Shuker et al., Science, 274: 1531-1534 (1996)] and smaller helicase fragments have not been previously reported. The 451 residue HCV NS3 helicase, which is about 48.2 kDa, is simply too large for effective use in such methods. Furthermore, to be useful, a fragment should be folded correctly, soluble, monodisperse, and stable in a buffered aqueous solution close to physiological conditions (pH 4-8 and salt concentrations less than about 250 mM). Therefore, it would be advantageous to have fragments of HCV NS3 helicase that are suitable for the most advanced techniques for characterizing proteins and designing inhibitors such as NMR, X-ray crystallography and ATPase assays such as the continuous spectrometric assay [Pullman et al., J Biol Chem, 235: 3322-3329 (1960)]. In addition, such fragments should be suitable for probing NTP and nucleic acid binding sites of the HCV NS3 helicase by NMR and crystallography, which together with mechanistic studies will provide insights into the mode of unwinding for HCV helicase.